Use of medications with neuroprotective properties to prevent or reduce the risk of ischemia-reperfusion injury in a subject

ABSTRACT

Provided are methods for treatment and prevention of ischemia-reperfusion injury and chronic intermittent hypoxia related injury through administering a neuroprotective compound. A subject benefiting from the method of the invention may be prescribed or undergoing anti-VEGF treatment, for example an IVAV treatment regimen, or may be diagnosed with a disorder such as sleep apnea.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.provisional application No. 62/239,639, filed Oct. 9, 2015, thedisclosures of which are incorporated herein by reference in theirentirety

BACKGROUND OF THE INVENTION

The eye, like other parts of the central nervous system, has limitedregeneration capability. Thus, many ocular diseases and injuries such asretinal photic injury, retinal ischemia-reperfusion induced eye injury,chronic intermittent hypoxia related eye injury, age-related maculardegeneration (AMD), and free-radical-mediated diseases are difficult totreat. AMD affects as many as 15 million Americans, with 200,000 newcases each year. Of these, approximately 10-15% further developexudative disease. Intravitreal anti-VEGF injection (IVAV) hasrevolutionized the treatment of exudative AMD. While AMD was the first,and is still the most common, indication for intravitreal anti-VEGFinjections, additional approved indications include central and branchretinal vein occlusion-related macular edema as well diabetic macularedema. These agents may also be used to diminish the effects ofproliferative diabetic retinopathy, vitreous hemorrhage, neovascularglaucoma, retinopathy of prematurity, choroidal neovascularization andmany other retinovascular diseases. However, IVAV treatment exposessubjects to risk for optic nerve injury and retinal nerve fiber layer(RNFL) loss caused by several mechanisms including decreasedneuroprotection (Chauhan et al., Invest Ophthalmol Vis Sci. 2002,43:2969-76; Weinreb and Khaw, Lancet 2004, 363:1711-1720; Michelson etal., Graefes Arch Clin Exp Ophthalmol. 1998, 236:80-5; Bonomi et al.,Ophthalmology, 2000, 107:1287-93; Kaur et al., ClinOphthalmol. 2008,2:879-89; Moore D et al., Clin Ophthalmol, 2008, 2:849-61; Leung et al.,Br J Ophthalmol, 2009, 93:964-8; Nishijima et al., Am J Pathol, 2007,171:53-67) and transient ischemia-reperfusion injury. The latter resultscumulatively in multiple brief episodes of tissue hypoxia similar tothose which occur as a result of chronic intermittent hypoxia (CIH)another cause of RNFL loss. Further, an IVAV treatment regimen generallyconsists of multiple IVAV injections leading to an increase incumulative risk for RNFL thinning or loss, as well as other risks suchas endophthalmitis, intraocular inflammation, hemorrhage, retinal tearor detachment, retinal vascular occlusion, blindness (Falavarjani etal., Eye, 2013, 27:787-94) and an increase in financial cost. Therefore,there is a need in the art for methods of protecting subjects undergoingVEGF treatments such as IVAV from secondary injury, as well as methodsfor reducing the total number of IVAV injections required for aneffective IVAV treatment regimen. The current invention satisfies thisneed.

SUMMARY OF THE INVENTION

In one embodiment, the invention relates to a method of protectingagainst ischemia-reperfusion injury in a subject in need thereof,comprising administering a neuroprotective compound to the subject,wherein the subject is selected from the group consisting of anon-glaucomatous subject and a glaucomatous subject not using aneuroprotective compound.

In one embodiment, a subject will experience or has been diagnosed withone or more of a medical condition or a procedure associated with riskof ischemia-reperfusion injury or chronic intermittent hypoxia. In oneembodiment, the condition or procedure is selected from the groupconsisting of retinal ischemia-induced eye injury, age-related maculardegeneration, sleep apnea, diabetes, transient ischemic attack,cardiovascular surgery, and cardiac arrest.

In one embodiment, the neuroprotective compound is capable of crossingthe blood-brain barrier and the blood-retina barrier. In one embodiment,the compound is administered to the subject by way of an administrationroute selected from the group consisting of topical, oral andintravenous. In one embodiment, the compound is a therapeutic compound.In one embodiment, the compound is tafluprost.

In one embodiment, the neuroprotective compound is administered to thesubject daily for at least three months.

In one embodiment, a subject is further in need of or has beenadministered an anti-VEGF treatment. In one embodiment, the compound isadministered to the subject by way of one or more treatment regimenselected from the group consisting of at least once prior to anti-VEGFtreatment and at least once following anti-VEGF treatment.

In one embodiment, a subject will experience or has experienced aprocedure associated with risk of ischemia-reperfusion injury. In oneembodiment, the subject will experience or has experienced IVAV. In oneembodiment, a compound is administered to the subject by way of one ormore administration route selected from the group consisting of topical,oral, intravenous, periocular injection and an intraocular implant drugdelivery device. In one embodiment, the compound is administered to thesubject daily for the duration and for at least three months followingan IVAV treatment regimen. In one embodiment, the method of theinvention allows that one or more of the time interval betweeninjections is increased and the number of injections is decreasedrelative to the average time interval and number of injections receivedrespectively by control individuals.

In one embodiment, the invention relates to a method of treating orprotecting a subject from RNFL thinning or loss and ischemia-reperfusioninjury associated with IVAV treatment, the method comprising performingintraoperative paracentesis on the subject, when the subject has notreceived a neuroprotective compound prior to receiving an IVAVinjection. In one embodiment, the method further comprises administeringa neuroprotective compound to the subject at least once following anIVAV injection. In one embodiment, the compound is administered to thesubject by way of one or more administration route selected from thegroup consisting of topical, oral, intravenous, periocular injection andan intraocular implant drug delivery device. In one embodiment, thecompound is administered to the subject daily for the duration of anIVAV treatment regimen. In one embodiment, the compound is tafluprost.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1 depicts baseline characteristics of eyes prior to IVAV treatment.Continuous data are presented as mean (standard deviation). Superscript¹indicates use of ANOVA; superscript² indicates use of Chi-square test.

FIG. 2 depicts post-injection data from follow-up examination, anaverage of 29 months post-injection. Continuous data are presented asmean (standard deviation). Superscript¹ indicates use of ANOVA.

FIG. 3 depicts the rate change in RNFL thinning. Continuous data arepresented as mean (standard deviation). Superscript¹ indicates use ofANOVA.

FIG. 4 depicts data showing a correlation between immediate postinjection IOP and change in RNFL thinning or loss.

DETAILED DESCRIPTION

The present invention relates to a method of preventing or reducing therisk of ischemia-reperfusion injury in an individual receiving or aboutto receive an anti-VEGF treatment. In one embodiment, the presentinvention provides a safer and more effective way to treat ophthalmicdisease and to perform ophthalmic surgeries. In one embodiment, theinvention relates to a method of prophylaxis and treatment of opticnerve injury and RNFL thinning or loss in a subject. In one embodiment,the invention relates to a method for increasing the interval betweenIVAV injections during an IVAV treatment regimen. In one embodiment, theinvention relates to an IVAV treatment regimen having a reduced numberof IVAV injections.

In one embodiment, the invention relates to a method for preventing orreducing optic nerve injury and RNFL thinning or loss in a subjectassociated with one or more of a degenerative eye condition, anophthalmic disease and an ophthalmic procedure by administering to thesubject a neuroprotective measure or a therapeutically effective amountof a neuroprotective compound. Ophthalmic diseases and degenerative eyeconditions that may be treated using the method of the inventioninclude, but are not limited to macular degeneration, includingage-related macular degeneration (AMD) and exudative or wet AMD, macularedema secondary to retinal vascular disease including diabeticretinopathy and retinal vein occlusions and intraocular inflammation;various forms of ocular neovascularization including cornealneovascularization, rubeosis, iris neovascularization, neovascularglaucoma, proliferative retinopathy, radiation retinopathy, retinopathyof prematurity, retinal angiomatous proliferans, Coats' disease, Eales'disease, and choroidal neovascularization secondary to numerous causesincluding uveitis, parafoveal telangectasis, angoid streaks, choroidalrupture, myopia. In one embodiment, the method of the invention maymodulate ocular wound healing including following ocular surgery such astrabeculectomy.

In one embodiment, the method of the invention involves administering aneuroprotective compound to a subject in need of or having undergone ananti-VEGF treatment. In one embodiment, the invention relates to amethod for reducing the optic nerve injury and RNFL thinning or lossassociated with IVAV treatment in a subject, by administering to thesubject a neuroprotective measure or a therapeutically effective amountof a neuroprotective compound. In one embodiment, a neuroprotectivecompound is pharmaceutical used for treatment of glaucoma.

In one embodiment, the method of the invention relates to performingintraoperative paracentesis during IVAV as a neuroprotective measure.

Definitions

As used herein, each of the following terms has the meaning associatedwith it in this section.

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the clinical procedures are thosewell-known and commonly employed in the art.

As used herein, the articles “a” and “an” refer to one or to more thanone (i.e. to at least one) of the grammatical object of the article. Byway of example, “an element” means one element or more than one element.

As used herein, the term “about” will be understood by persons ofordinary skill in the art and will vary to some extent on the context inwhich it is used. As used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, the term “about”is meant to encompass variations of ±20% or ±10%, more preferably ±5%,even more preferably ±1%, and still more preferably ±0.1% from thespecified value, as such variations are appropriate to perform thedisclosed methods.

The term “abnormal” when used in the context of organisms, tissues,cells or components thereof, refers to those organisms, tissues, cellsor components thereof that differ in at least one observable ordetectable characteristic (e.g., age, treatment, time of day, etc.) fromthose organisms, tissues, cells or components thereof that display the“normal” (expected) respective characteristic. Characteristics which arenormal or expected for one cell or tissue type, might be abnormal for adifferent cell or tissue type.

As used herein, “autologous” refers to a biological material derivedfrom the same individual into whom the material will later bere-introduced.

As used herein, “allogenic” refers to a biological material derived froma genetically different individual of the same species as the individualinto whom the material will be introduced.

A disease or disorder is “alleviated” if the severity of a symptom ofthe disease or disorder, the frequency with which such a symptom isexperienced by a subject, or both, is reduced.

As used herein, a “disease” is a state of health of a subject whereinthe subject cannot maintain homeostasis, and wherein if the disease isnot ameliorated then the subject's health continues to deteriorate.

As used herein, a “disorder” in a subject is a state of health in whichthe subjecl is able to maintain homeostasis, but in which the subject'sstate of health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the subject's state of health.

As used herein, the term “prevent” or “prevention” means no disorder ordisease development if none had occurred, or no further disorder ordisease development if there had already been development of thedisorder or disease. Also considered is the ability of one to preventsome or all of the symptoms associated with the disorder or disease.

As used herein, the terms “effective amount,” “pharmaceuticallyeffective amount” and “therapeutically effective amount” refer to anontoxic but sufficient amount of an agent to provide the desiredbiological result. That result can be reduction and/or alleviation ofthe frequency and/or severity of signs, symptoms, or causes of adisease, or any other desired alteration of a biological system. Anappropriate therapeutic amount in any individual case may be determinedby one of ordinary skill in the art using routine experimentation.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human.

As used herein, the term “pharmaceutically acceptable” refers to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the compound, and is relativelynontoxic, i.e., the material may be administered to an individualwithout causing undesirable biological effects or interacting in adeleterious manner with any of the components of the composition inwhich it is contained.

As used herein, the term “pharmaceutical composition” refers to amixture of at least one compound of the invention with other chemicalcomponents, such as carriers, stabilizers, diluents, dispersing agents,suspending agents, thickening agents, and/or excipients. Thepharmaceutical composition facilitates administration of the compound toan organism. Multiple techniques of administering a compound exist inthe art including, but not limited to: intravenous, oral, aerosol,parenteral, ophthalmic, pulmonary and topical administration.

As used herein, the term “pharmaceutically acceptable carrier” means apharmaceutically acceptable material, composition or carrier, such as aliquid or solid filler, stabilizer, dispersing agent, suspending agent,diluent, excipient, thickening agent, solvent or encapsulating material,involved in carrying or transporting a compound useful within theinvention within or to the subject such that it may perform its intendedfunction. Typically, such constructs are carried or transported from oneorgan, or portion of the body, to another organ, or portion of the body.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation, including the compound usefulwithin the invention, and not injurious to the subject.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression,diminution, remission, or eradication of at least one sign or symptom ofa disease or disorder state.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Description

IVAV, the standard of care associated with multiple ocular diseases,causes an immediate, transient elevation of intraocular pressure (IOP)which temporarily compromises intraocular circulation. The resultanttransient ischemia-reperfusion episodes result in ischemic tissue injurysimilar to that which occurs in sleep apnea, a condition characterizedby chronic intermittent hypoxia. The current invention is based on thediscovery that VEGF suppression, intended to modulate detrimentalangiogenic and vasopermiability effects, also chronically compromisesVEGF's physiologic neuroprotective function rendering eyes moresusceptible to ischemia-reperfusion injury.

The present invention is based upon the surprising discovery thatneuroprotective measures can be used prophylactically andtherapeutically in prevention of RNFL thinning or loss and/or ocularischemia-reperfusion injury associated with intravitreal anti-VEGFtreatment. Specifically, both intraoperative paracentesis andadministration of neuroprotective anti-glaucoma medications were foundto reduce the RNFL thinning or loss when administered to patientsreceiving IVAV.

In one embodiment, the invention relates to the use of neuroprotectivecompounds prophylactically and therapeutically in prevention ofischemia-reperfusion injury in a subject undergoing or in need of ananti-VEGF treatment regimen. In one embodiment, the invention relates tothe use of neuroprotective compounds prophylactically andtherapeutically in prevention of tissue injury due to chronicintermittent hypoxia.

In one embodiment, a neuroprotective measure comprises providing atreatment regimen of a neuroprotective compound to a subject at risk ofone or more of RNFL thinning or loss and/or ischemia-reperfusion injury.In one embodiment, a neuroprotective compound is an anti-glaucomamedication. In one embodiment, a neuroprotective measure comprisesperforming paracentesis. In one embodiment, a combination ofparacentesis and a neuroprotective compound is administered to a subjectat risk of one or more of RNFL thinning or loss and/or ocularischemia-reperfusion injury.

Use

A risk of ischemia-reperfusion injury is often associated with vascularand cardiac surgery, but can also be a concern in transplantationsurgery or a complication of disease such as diabetes. A similarintermittent tissue injury due to intermittent ischemia may also occurin persons with chronic intermittent hypoxemia due to sleep apnea. Thecurrent invention identifies individuals undergoing an anti-VEGFtreatment as having an increased risk of complications arising fromintermittent tissue ischemia due to ischemia-reperfusion injury, howeverany individual at risk of intermittent tissue ischemia due toischemia-reperfusion injury or CIH can benefit from the method of theinvention.

Neuroprotective compounds, including but not limited to anti-glaucomamedications, provide a protective measure to reduce the risk ofcomplications arising from ischemic tissue injury. Therefore, in oneembodiment, the method of the invention relates to administering aneuroprotective compound to a subject at risk of ischemia-reperfusioninjury or CIH. In one embodiment, the subject is about to receive orreceiving an anti-VEGF medication. In one embodiment, the method relatesto administering a neuroprotective compound to a subject receiving ananti-VEGF medication prior to, concurrent with, or following a procedureor condition associated with risk of ischemia-reperfusion injury orchronic intermittent hypoxia.

In one embodiment, a procedure associated with risk ofischemia-reperfusion injury is an injection. In one embodiment, aninjection is an intravitreal injection. In one embodiment, a procedureassociated with risk of ischemia-reperfusion injury is a surgicalprocedure. Non-limiting examples of surgical procedures that areassociated with risk of ischemia-reperfusion injury includecardiovascular surgery, liver transplantation surgery, vitrectomy, freeautologous tissue transfer, flap surgery, and vascular surgery.

In one embodiment, a procedure associated with risk ofischemia-reperfusion injury and/or CIH is continuous positive airwaypressure (CPAP), in an individual having sleep apnea.

In one embodiment, a procedure associated with risk ofischemia-reperfusion injury is a non-medical procedure. In oneembodiment, a non-medical procedure associated with risk ofischemia-reperfusion injury is sleep, in a subject having sleep apnea.In one embodiment, a non-medical procedure associated with risk ofischemia-reperfusion injury may include routine or daily activities suchas walking, in a subject having diabetes.

In one embodiment, the neuroprotective compound of the invention isprovided to a subject receiving or about to receive an anti-VEGFmedication. Such a subject may be diagnosed with any disease for whichan anti-VEGF medication is indicated. In one embodiment, a subject isdiagnosed as having cancer.

In one embodiment, a subject receiving or about to receive an anti-VEGFmedication is a subject having an ophthalmic disease or degenerative eyecondition. In one embodiment, an ophthalmic diseases or degenerative eyecondition is one of macular degeneration, including age-related maculardegeneration (AMD) and exudative or wet AMD, macular edema secondary toretinal vascular disease including diabetic retinopathy and retinal veinocclusions and intraocular inflammation; various forms of ocularneovascularization including corneal neovascularization, rubeosis, irisneovascularization, neovascular glaucoma, proliferative retinopathy,radiation retinopathy, retinopathy of prematurity, retinal angiomatousproliferans, Coats' disease, Eales' disease, and choroidalneovascularization secondary to numerous causes including uveitis,parafoveal telangectasis, angoid streaks, choroidal rupture and myopia.

In one embodiment, a subject receiving or about to receive an anti-VEGFmedication is at risk of ischemia-reperfusion injury. In one embodiment,a subject receiving or about to receive an anti-VEGF medication is atrisk of an ischemia-reperfusion injury associated pathology. In oneembodiment, a subject receiving or about to receive an anti-VEGFmedication is at risk of RNFL thinning or loss due toischemia-reperfusion injury.

In one embodiment, a subject receiving or about to receive an anti-VEGFmedication at risk of RNFL thinning or loss due to ischemia-reperfusioninjury or chronic intermittent hypoxia is a subject not having beendiagnosed with glaucoma or not taking a neuroprotective anti-glaucomamedication at the time an anti-VEGF agent or treatment is prescribed orscheduled.

In one embodiment, a neuroprotective measure is administered to asubject about to receive or having received a treatment comprising ananti-VEGF agent. Anti-VEGF agents include, but are not limited to,Bevacizumab (e.g., Avastin®, Genentech/Roche, Inc., South San Francisco,Calif.), Ranibizumab (e.g., Lucentis®, Genentech/Roche, Inc., South SanFrancisco, Calif.), Pegaptanib (e.g., Macugen®, Eyetech, Inc., CedarKnolls, N.J.), aflibercept (e.g., Eylea®, Regeneron Pharmaceuticals,Inc., Tarrytown, N.Y.), brolucizumab (Alcon), Conbercept (Lumitin,Chengdu Kang Hong Biotech, approved in China), NT-503 (Neurotech),dexamethasone, an intravitreal implant (e.g., Ozurdex®, Allergan, Inc.,Irvine, Calif.), Anecortave acetate, VEGF-trap, Lapatinib (Tykerb),Sorafenib (Nexavar), Sunitinib (Sutent), Axitinib, and Pazopanib,Squalamine lactate, Combretastatin A4 Prodrug, AdPEDF, SiRNA, Cand5, andTG100801.

In one embodiment, the neuroprotective compound of the invention isprovided to a subject at risk of ischemia-reperfusion injury or chronicintermittent hypoxia. Such a subject may be diagnosed with diabetes,sleep apnea, transient ischemic attack or a disease requiring one ormore of cardiovascular surgery, liver transplantation surgery,vitrectomy, free autologous tissue transfer, flap surgery, and vascularsurgery.

In one embodiment, a subject at risk of ischemia-reperfusion injury orchronic intermittent hypoxia is a subject not having been diagnosed withglaucoma or not taking a neuroprotective anti-glaucoma medication.

In one embodiment, ischemia-reperfusion injury and chronic intermittenthypoxia related injury may occur in any tissue or organ. Organs that aresusceptible to ischemia-reperfusion injury and chronic intermittenthypoxia related injury include but are not limited to the eye, heart,liver and brain. Thus, any tissue, organ, or organ system may benefitfrom protection from ischemia-reperfusion injury and chronicintermittent hypoxia related injury using the method of the invention.

Treatment Regimen

The invention is based on the discovery that patients diagnosed ashaving glaucoma and taking an anti-glaucoma medication prior andsubsequent to IVAV treatment had a reduced level of RNFL thinning orloss as compared to patients not using anti-glaucoma medications.Therefore, one aspect of the invention relates to a method of treatingor preventing RNFL thinning or loss in a non-glaucomatous individual inneed of IVAV through administering to the individual a treatment regimenof a neuroprotective anti-glaucoma medication. In one embodiment, aneuroprotective compound is administered prior to IVAV treatment,subsequent to IVAV treatment or both. In one embodiment, a treatmentregimen comprises administering a neuroprotective compound at least oncedaily for at least 2, at least 3, at least 4, at least 5 or more daysprior to an IVAV procedure. In one embodiment, a treatment regimencomprises administering a neuroprotective compound at least once dailyfor at least 1 day, at least 7 days, at least 10 days, at least 1 month,at least 2 months, at least 3 months, at least 6 months, at least 1 yearor more than 1 year following an individual IVAV procedure. In oneembodiment, a treatment regimen comprises administering aneuroprotective compound at least once daily for at least 1 day, atleast 7 days, at least 10 days, at least 1 month, at least 2 months, atleast 3 months, at least 6 months, at least 1 year or more than 1 yearfollowing each individual IVAV injection throughout the duration of anIVAV treatment regimen. In one embodiment, a treatment regimen comprisesadministering a neuroprotective compound at least once daily for atleast 2, at least 3, at least 4, at least 5 or more days prior to anIVAV procedure and for at least 1 day, at least 7 days, at least 10days, at least 1 month, at least 2 months, at least 3 months, at least 6months, at least 1 year or more than 1 year following an individual IVAVprocedure. In one embodiment, a treatment regimen comprisesadministering a neuroprotective compound at least once daily for atleast 2, at least 3, at least 4, at least 5 or more days prior to anIVAV procedure and for at least 1 day, at least 7 days, at least 10days, at least 1 month, at least 2 months, at least 3 months, at least 6months, at least 1 year or more than 1 year following each individualIVAV injection throughout the duration of a IVAV treatment regimen.

Most IVAV treatment regimens include multiple IVAV procedures spacedover several months, a year, or even several years. Therefore, in oneembodiment, a treatment regimen includes administering a neuroprotectivecompound to a non-glaucomatous subject in need of IVAV treatment dailyduring the whole treatment period for multiple IVAV procedures. Further,the administration of the neuroprotective compound of the invention hasa benefit in reducing the duration of IVAV treatment or the number ofIVAV procedures necessary for treating an ocular condition.

In one embodiment, a neuroprotective measure is administered to asubject about to experience or having experienced IVAV. In oneembodiment, the invention relates to therapeutic use of neuroprotectivemeasure for a subject in need of IVAV both prior to and following thesurgical procedure (e.g. a daily eye-drop or medication for use prior toand following the procedure). In one embodiment, the invention relatesto therapeutic use of neuroprotective measure for a subject in need ofIVAV during the surgical procedure (e.g. paracentesis). In oneembodiment, the invention relates to therapeutic use of neuroprotectivemeasure for a subject in need of IVAV during and following the procedure(e.g. a combination of paracentesis and a daily eye-drop or medicationfor use following the procedure). In one embodiment, administration ofthe neuroprotective compound of the invention has a benefit in reducingone or more of the duration of IVAV treatment and the number of IVAVprocedures necessary for treating an ocular condition.

In one embodiment, a neuroprotective compound is administered for atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 days prior toadministration of an anti-VEGF agent. In one embodiment, aneuroprotective compound is administered daily for at least 1 day, atleast 7 days, at least 10 days, at least 1 month, at least 2 months, atleast 3 months, at least 6 months, at least 1 year or more than 1 yearfollowing administration of an anti-VEGF agent. In one embodiment, aneuroprotective compound is administered chronically.

In one embodiment, a neuroprotective compound is administered to asubject taking an anti-VEGF agent for at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more than 10 days prior to a surgical procedure. In oneembodiment, a neuroprotective compound is administered to a subjecttaking an anti-VEGF agent daily for at least 1 day, at least 7 days, atleast 10 days, at least 1 month, at least 2 months, at least 3 months,at least 6 months, at least 1 year or more than 1 year following asurgical procedure. In one embodiment, a neuroprotective compound isadministered chronically.

In one embodiment, a neuroprotective measure is used in combination witha neuroprotective compound. In one embodiment, intraoperativeparacentesis is used in combination with a treatment regimen comprisinga neuroprotective compound. Such a combination may be useful inprotecting a subject from RNFL thinning or loss when the subject has notbeen administered a neuroprotective compound prior to receiving IVAV. Inone embodiment, a subject who has not been administered aneuroprotective compound is a subject who has not been provided with orwho has not taken a neuroprotective compound at least once daily for atleast 2, at least 3, at least 4, at least 5 or more days prior to anIVAV procedure.

Treat and Extend

The treat-and-extend strategy (T&E) generally begins with monthlyloading injection(s)—generally 1-3—after which the patient returns in 4weeks and is evaluated for signs of disease activity. The patientreceives an injection and if the disease is active, returns in 4 weeksfor another; if no evidence of disease activity is seen, the patientreturns in 5 or 6 weeks. The patient receives an injection at each visitwith the results of the examination determining the interval to the nextvisit. If inactive, the interval is extended by 1 or 2 weeks; if active,the interval is decreased by 2 weeks and then kept constant. Generally,once the optimal treatment interval is determined for a given eye, itusually remains constant.

In one embodiment, the method of the invention is used with a T&Estrategy to one or more of reduce the total number of IVAV injectionsthat an individual receives and increase the interval between IVAVinjections. In one embodiment, the method reduces the number of initialmonthly loading IVAV injections. In one embodiment, the method allowsfor a higher percentage of patients to be extended to the maximumrecommended interval between IVAV injections (generally 10-12 weeks).

Compounds

Neuroprotective compounds that may be administered according to theinvention include, but are not limited to, acetazolamide, alphaagonists, betaxolol, bimatoprost 0.01% ophthalmic solution, bimatoprost0.03% ophthalmic solution, brimonidine, brinzolaminde, a combination ofbrimonidine plus brinzolamide, Ca2+ channel blockers, carbonic anhydrideinhibitors, chondroitin sulfate proteoglycan, dorzolamide, ginkgo bilobaextract, latanoprost, L-glutamate, memantine, neurotrophins, nipradilol,Nitric Oxide Synthase inhibitors, NMDA antagonists, phenylephrine,prostaglandin analogues, tafluprost, timolol, timolol XE, a combinationof timolol plus brimonidine, travoprost 0.004% eye drop, travoprost0.004% eye drop-Travatan Z and unoprostone isopropyl ophthalmicsolution, nalmefene, minocycline, recombinant human basic fibroblastgrowth factor (bFGF), citicoline, green tea extract and epoetin alfa. Inone embodiment, a neuroprotective compound is an anti-glaucomamedication.

In one embodiment, a composition comprising a neuroprotective compoundis preservative free. In one embodiment, a preservative freeneuroprotective compound is tafluprost.

In another embodiment of the invention, a therapeutically effectiveamount of a neuroprotective compound is used in combination therapy witha broad range of presently available ocular therapeutics. Hence, aneuroprotective compound can be coadministered with at least one othertherapeutically active agent in the same delivery vehicle/carrier eithertopically, orally, or intravenously. Alternatively, the combinationtherapy can be coadministered using separate routes and dosage forms,(e.g., neuroprotective eye drop and oral antibiotic). Within the scopeof the invention, a neuroprotective measure or compound is efficaciouslycombined with at least one of an antibiotic (e.g., beta-lactam type,fluoroquinolones, peptide antibiotics, broad-spectrum penicillins,fortified antibiotic mixtures), an antibacterial, a free-radicalscavenging antioxidant, an antiviral, a corticosteroid, a non-steroidalanti-inflammatory, a cycloplegic, a cholinergic, an aqueous or salineirrigating solution, a miotic, a collagenase inhibitor, a carbonicanhydrase inhibitor, a glycoprotein, a growth factor, silver nitrate,and an ocular tissue adhesive/corneal mortar (for acutely inflamedperforated corneas).

In one embodiment, a neuroprotective measure or compound can becoadministered with a therapeutically effective amount of an antibioticexemplified by ciprofloxacin, ofloxacin, norfloxacin, cefazolin,tobramycin, gentamycin, an aminoglycoside, a penicillin, a semisyntheticpenicillin, amoxicillin, ampicillin, carbenicillin, ticarcillin,mezlocillin, a cephalosporin, vancomycin, chloramphenicol, erythromycin,clindamycin, rifampin, bacitracin, polymyxin, spectinomycin, asulfonamide; and trimethoprim; a free-radical scavenging antioxidantexemplified by super oxide dismutase, a carotenoid (such as astaxanthin,canthazanthin, β-carotene, zeaxanthin, lutein and α-tocopherol),ascorbic acid, glutathione, selenous acid or sodium selenate, andcertain aminosteroids (e.g., as disclosed in U.S. Pat. No. 5,209,926);an antiviral exemplified by acyclovir, ganciclovir, idoxuridine,vidarabine, trifluridine, bromovinyldeoxyuridine, azidothymidine,amantadine, rimantadine; a corticosteroid exemplified by dexamethasone,prednisolone, prednisone, fluorometholone, betamethasone,hydrocortisone; an non-steroidal antiinflammatory agent exemplified byketorolac, indomethacin, flurbiprofen, ketoprofen, loxoprofen, anddiclofenac; a cycloplegic exemplified by atropine; a moitic exemplifiedby physostigmine, pilocarpine, and carbachol; a collagenase inhibitorexemplified by acetyl cysteine; a glycoprotein such as fibronectin andvitronectin, as well as analogs or fragments thereof, an ocular tissueadhesive as exemplified by isobutyl cyanoacrylate; a corneal mortarexemplified by fibronectin/growth factor (e.g., EGF) composition,optionally with a protein crosslinking agent (e.g., aldehydes anddi-imidate esters); and various admixtures of the above materials.

The precise neuroprotective measure or prophylactic or therapeuticdosage of a neuroprotective compound to be employed depends upon severalfactors, including the age and physical condition of the subject, thenature and the severity of the ocular condition being treated, and theroute of dosage administration. The assessment of these factors as wellas the determination of the precise dosage is well within the skill ofthe treating ophthalmologist. In one embodiment, a neuroprotectivecompound is administered in an amount or at a dose that does not resultin substantial toxicity to the eye. As used herein, a lack ofsubstantial toxicity encompasses both the absence of any manifestationsof toxicity, as well as manifestations of toxicity that one skilled inthe art would consider not sufficiently detrimental to decrease or ceasetreatment.

Formulations and Administration

Administration of the neuroprotective measures or compounds of thepresent invention to a subject, preferably a mammal, more preferably ahuman, may be carried out using known procedures, at dosages and forperiods of time effective to prevent or treat one or more of ischemictissue injury due to ischemia-reperfusion injury or CIH, and RNFLthinning or loss in the subject. An effective amount of the therapeuticcompound necessary to achieve a therapeutic effect may vary according tofactors such as the state of the disease or disorder in the subject; theage, sex, and weight of the subject; and the ability of the therapeuticcompound to cross the blood-brain barrier and the blood-retina barrier

The regimen of administration may affect what constitutes an effectiveamount. The neuroprotective measure or compounds may be administered tothe subject one or more of prior to, during and after IVAV treatment orthe administration of an anti-VEGF agent. Further, the dosages of theneuroprotective compounds may be proportionally increased or decreasedas indicated by the exigencies of the therapeutic or prophylacticsituation. A non-limiting example of an effective dose range for atherapeutic compound of the invention is from about 1 and 5,000 mg/kg ofbody weight/per day. One of ordinary skill in the art would be able tostudy the relevant factors and make the determination regarding theeffective amount of the therapeutic compound without undueexperimentation.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular subject, composition, and mode ofadministration, without being toxic to the subject.

In particular, the selected dosage level will depend upon a variety offactors including the activity of the particular compound employed, thetime of administration, the rate of excretion of the compound, theduration of the treatment, other drugs, compounds or materials used incombination with the compound, the age, sex, weight, condition, generalhealth and prior medical history of the subject being treated, and likefactors well, known in the medical arts.

A medical doctor, e.g., physician or veterinarian, having ordinary skillin the art may readily determine and prescribe the effective amount ofthe pharmaceutical composition required. For example, the physician orveterinarian could start doses of the compounds of the inventionemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.

Compounds of the invention for administration may be in the range offrom about 1 μg to about 10,000 mg, about 20 μg to about 9,500 mg, about40 μg to about 9,000 mg, about 75 μg to about 8,500 mg, about 150 μg toabout 7,500 mg, about 200 μg to about 7,000 mg, about 3050 μg to about6,000 mg, about 500 μg to about 5,000 mg, about 750 μg to about 4,000mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 50 mg toabout 1,000 mg, about 75 mg to about 900 mg, about 100 mg to about 800mg, about 250 mg to about 750 mg, about 300 mg to about 600 mg, about400 mg to about 500 mg, and any and all whole or partial incrementstherebetween.

In some embodiments, the dose of a compound of the invention is fromabout 1 mg and about 2,500 mg. In some embodiments, a dose of a compoundof the invention used in compositions described herein is less thanabout 10,000 mg, or less than about 8,000 mg, or less than about 6,000mg, or less than about 5,000 mg, or less than about 3,000 mg, or lessthan about 2,000 mg, or less than about 1,000 mg, or less than about 500mg, or less than about 200 mg, or less than about 50 mg. Similarly, insome embodiments, a dose of a second compound (i.e., an apicomplexanparasite anti-infective) as described herein is less than about 1,000mg, or less than about 800 mg, or less than about 600 mg, or less thanabout 500 mg, or less than about 400 mg, or less than about 300 mg, orless than about 200 mg, or less than about 100 mg, or less than about 50mg, or less than about 40 mg, or less than about 30 mg, or less thanabout 25 mg, or less than about 20 mg, or less than about 15 mg, or lessthan about 10 mg, or less than about 5 mg, or less than about 2 mg, orless than about 1 mg, or less than about 0.5 mg, and any and all wholeor partial increments therebetween.

Eye drops and intravitreal or periocular (Sub-Tenon or subconjunctival)injections are the conventional dosage forms that account for greaterthan ninety percent of the currently available ophthalmic formulations.To improve bioavailability and reduce the complications associated withrepeated injections, the present invention contemplates sustaineddelivery of neuroprotective compounds alone or in combination with othermedications. The sustained delivery in the present invention can beachieved through a number of different delivery systems, including butnot limited to polymeric gels, colloidal systems including liposomes andnanoparticles, cyclodextrins, collagen shields, diffusion chambers,flexible carrier strips, and intravitreal implants.

In the present invention, ocular and intraocular drug delivery systemsdeliver these neuroprotective compounds to the back of the eye. Thesedrug delivery systems include: using the sclera itself as a drugdelivery reservoir, “prodrug” formulations that pass through the tissue,tiny biodegradable pellets that release the combinations over time,intravitreal implants, intravitreal silicone inserts, intravitreal andtransscleral poly(lactic-co-glycolic acid) microspheres,calcium-alginate inserts, encapsulated cells, transscleraliontophoresis, nanoparticles (e.g., calcium phosphate), and geneticallymodified viruses that can deliver therapeutic proteins into therapy.

Neuroprotective compounds of the present invention can be in the form ofsolutions. Solutions can be administered topically by applying them tothe cul-de-sac of the eye from a dropper controlled bottle or dispenser.A typical dose regimen for an adult human may range from about 2 toabout 8 drops per day, applied at bed-time or throughout the day.Dosages for adult humans may, however, be higher, in which case thedrops are administered by “bunching”, e.g., 5 doses administered over a5 minute period, repeated about 4 times daily. A topical solution inaccordance with one embodiment of the invention comprises a therapeuticdose of a neuroprotective compound in an artificial tear formulation.Such artificial tear formulations are used for restoring the normalbarrier function of damaged corneal epithelium following surgery.Typically, artificial tear compositions contain ionic components foundin normal human tear film, as well as various combinations of one ormore of tonicity agents (e.g., soluble salts, such as Na, Ca, K, and Mgchlorides, and dextrose and sorbitol), buffers (e.g., alkali metalphosphate buffers), viscosity/lubricating agents (e.g., alkyl andhydroxyalkyl celluloses, dextrans, polyacrylamides), nonionicsurfactants, sequestering agents (e.g., disodium edetate, citric acid,and sodium citrate), and preservatives (e.g., benzalkonium chloride, andthimerosal). In one embodiment, artificial tear compositions arepreservative free. The quantities and relative proportions of each ofthese components incorporated into an artificial tear composition arereadily determinable by the skilled formulation chemist. The ionicspecies bicarbonate is used in artificial tear compositions, e.g., U.S.Pat. No. 5,403,598 and Ubels, J L, et al, Arch. Ophthalmol. 1995, 113:371-8.

Alternatively, neuroprotective compounds of the present invention can bein the form of ophthalmic ointments. Ophthalmic ointments have thebenefit of providing prolonged drug contact time with the eye surface.Ophthalmic ointments will generally include a base comprised of, forexample, white petrolatum and mineral oil, often with anhydrous lanolin,polyethylene-mineral oil gel, and other substances recognized by theformulation chemist as being non-irritating to the eye, which permitdiffusion of the drug into the ocular fluid, and which retain activityof the medicament for a reasonable period of time under storageconditions.

Prophylactic and therapeutic amounts of a neuroprotective compound canbe administered orally. For these oral dosage forms, a neuroprotectivecompound may be formulated with a pharmaceutically acceptable solid orliquid carrier. Solid form preparations include powders, tablets, pills,capsules, cachets, and dispersible granules. The concentration oreffective amount of the neuroprotective compound to be administered perdosage is widely dependent on the actual compound. However, a total oraldaily dosage normally ranges from about 50 mg to 30 g, and morepreferably from about 250 mg to 25 g. A solid carrier can be one or moresubstances which may also function as a diluent, a flavoring agent, asolubilizer, a lubricant, a suspending agent, a binder, a preservative,a tablet disintegrating aid, or an encapsulating material. Suitablecarriers include magnesium carbonate, magnesium stearate, talc, sugar,lactose, pectin, dextrin, starch gelatin, tragacanth, methylcellulose,sodium carboxymethylcellulose, microcrystalline cellulose, a low meltingwax, cocoa butter, and the like. The term “preparation” is intended toinclude the formulation of the active compound with encapsulatingmaterial as a carrier providing a capsule in which the active component,with or without other carriers, is surrounded by a carrier, which isthus in association with it. Similarly, cachets and lozenges areincluded. Tablets, powders, capsules, pills cachets, and lozenges can beused as solid dosage forms suitable for oral administration.

A neuroprotective compound may also be administered surgically as anocular implant. As one example, a reservoir container having adiffusible wall of polyvinyl alcohol or polyvinyl acetate and containingmilligram quantities of a neuroprotective compound may be implanted inthe sclera. As another example, a neuroprotective compound in milligramquantities may be incorporated into a polymeric matrix having dimensionsof about 2 mm by 4 mm, and made of a polymer such as polycaprolactone,poly(glycolic) acid, poly(lactic) acid, or a polyanhydride, or a lipidsuch as sebacic acid, and may be implanted on the sclera or in the eye.This is usually accomplished with the subject receiving either a topicalor local anesthetic and using a small (3-4 mm incision) made behind thecornea. The matrix, containing a neuroprotective compound, is theninserted through the incision and sutured to the sclera using 9-0 nylon.

A neuroprotective compound may also be contained within an inert matrixfor either topical application or injection into the eye. As one exampleof an inert matrix, liposomes may be prepared from dipalmitoylphosphatidylcholine (DPPC), preferably prepared from eggphosphatidylcholine (PC) since this lipid has a low heat transition.Liposomes are made using standard procedures as known to one skilled inthe art. A neuroprotective compound, in amounts ranging from nanogram tomicrogram quantities, is added to a solution of egg PC, and thelipophilic drug binds to the liposome.

A time-release drug delivery system may be implanted intraocularly toresult in sustained release of the active agent over a period of time.The implantable formation may be in the form of a capsule of a polymer(e.g., polycaprolactone, poly(glycolic) acid, poly(lactic) acid,polyanhydride) or lipids that may be formulation as microspheres. As anillustrative example, a neuroprotective compound may be mixed withpolyvinyl alcohol (PVA), the mixture then dried and coated with ethylenevinyl acetate, then cooled again with PVA. The neuroprotective compoundbound with liposomes may be applied topically, either in the form ofdrops or as an aqueous based cream, or may be injected intraocularly. Ina formulation for topical application, the drug is slowly releasedovertime as the liposome capsule degrades due to wear and tear from theeye surface. In a formulation for intraocular injection, the liposomecapsule degrades due to cellular digestion. Both of these formulationsprovide advantages of a slow release drug delivery system, allowing thesubject a constant exposure to the drug over time.

In a time-release formulation, the microsphere, capsule, liposome, etc.may contain a concentration of a neuroprotective compound that could betoxic if administered as a bolus dose. The time-release administration,however, is formulated so that the concentration released at any periodof time does not exceed a toxic amount. This is accomplished, forexample, through various formulations of the vehicle (coated or uncoatedmicrosphere, coated or uncoated capsule, lipid or polymer components,unilamellar or multilamellar structure, and combinations of the above,etc.). Other variables may include the subject'spharmacokinetic-pharmacodynamic parameters (e.g., body mass, gender,plasma clearance rate, hepatic function, etc.). The formation andloading of microspheres, microcapsules, liposomes, etc. and their ocularimplantation are standard techniques known by one skilled in the art,for example, the use a ganciclovir sustained-release implant to treatcytomegalovirus retinitis, disclosed in Vitreoretinal SurgicalTechniques, Peyman et al., Eds. (Martin Dunitz. London 2001, chapter45); Handbook of Pharmaceutical Controlled Release Technology, Wise, Ed.(Marcel Dekker, New York 2000), the relevant sections of which areincorporated by reference herein in their entirety.

A further aspect of the invention is intraoperative paracentesis. Foradministration of intraoperative paracentesis, anesthesia may beadministered via methods known to those of skill in the art including,but not limited to, topical administration of proparacaine or tetracainedrops, 2% lidocaine gel or subconjunctival injection of 2% lidocainesolution. In general, intravitreal injections are administered undercontrolled aseptic conditions, using mask, sterile gloves, a steriledrape, and a sterile lid speculum. Prior to injection the perioculararea is cleaned. Solutions for use in cleaning the periocular area areknown to those of skill in the art and may include a povidone-iodinepreparation. Intraoperative paracentesis (PARA) may be performedimmediately following an intravitreal injection, such as IVAV. PARA maybe performed by methods known to those of skill in the art including,for example, using a 30 g needle on a tuberculin syringe from which theplunger had been removed. Following PARA, irrigation of the eyes may beperformed with an appropriate solution. In one embodiment, a solutionappropriate for use is a balanced salt solution.

EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Example 1: Effect of Intravitreal Anti-VEGF Injection Performed with andwithout Paracentesis on Retinal Nerve Fiber Layer Thickness

In a previous study, a trend towards RNFL thinning or loss in eyes thatreceived IVAV without adjunctive paracentesis (PARA) was observed (Haleet al., The Retina Society Annual Meeting, 2010). However, this studywas limited by retrospective design, small sample size, small number ofinjections and short follow-up period. The purpose of the present studyis to investigate the impact of IVAV performed with and without PARA onRNFL thinning or loss.

The materials and methods are now described.

Materials and Methods

Trial Design

This cohort study compared IVAV with and without PARA in two parallelgroups with AMD followed for at least 3 months. The study was performedat a single institution in northeastern Pennsylvania.

Study Population

The study population included all Geisinger Medical Center subjects whobegan treatment with IVAV between January 2008 and May 2014. Thescheduling staff, which was blinded to the study treatments, assignedeight hundred eighty three subjects to one of two retina specialists.One physician performed paracentesis on all subjects, while the otherdid not perform paracentesis on any subject. Number of potentialsubjects was smaller in the PARA subgroup because that physician workedfewer days per week at the study location. Each retina specialistalerted the Clinical Research Coordinator (CRC) about potential studyparticipants at the time of the initial exam. The CRC discussed thestudy with candidates and those who accepted the invitation toparticipate and who met inclusion criteria (168 subjects, 196 eyes) wereenrolled. Five eyes (3 subjects) with poor quality OCT due to mediaopacity or poor fixation, 8 eyes (8 subjects) with juxtapapillaryneovascularization and 4 eyes (4 subjects) with fewer than 3 monthsfollow up were excluded, resulting in 179 eyes of 153 subjects. TheGeisinger Institutional Review Board approved the study. Writteninformed consent was obtained from all participants and the study wasconducted in accordance with the Declaration of Helsinki. The study wasHIPAA compliant. All testing and treatment was performed in theGeisinger Medical Center Department of Ophthalmology in Danville, Pa.,USA.

Assessments

Ophthalmic photographers obtained OCT (Cirrus, Carl Zeiss Meditec, Inc.)prior to each injection and scanned images into the electronic medicalrecord. RNFL thickness was calculated by the autosegmentation algorithm.An ophthalmic nurse obtained intraocular pressure with tonopen beforeand immediately and 10 minutes after injection. Tonopen was used in lieuof applanation in order to facilitate immediate post-injection IOPmeasurement. If the 10-minute post injection IOP was greater than 30 mmhg, additional IOP readings were obtained at 10 minute intervals untilIOP was 30 mm hg or less.

Outcomes

The primary outcome measures were the mean change in RNFL thinning orloss (chgRNFL thinning or loss) between initial and final measurementsand the rate of RNFL thinning or loss change (rate chgRNFLT). Secondaryoutcomes calculated for each visit were the mean pre to post injectionchange of IOP (chgIOP) and the maximum post injection IOP (maxIOP) andfinal IOP. The highest maxIOP and the largest chgIOP recorded for eacheye throughout the course of treatment were used to compute mean values.IOP prior to injection at last visit was recorded as final IOP. Adverseevents monitored included endophthalmitis, hyphema, lens injury,pre-injection IOP>25.

Intervention

Intravitreal injections were performed using 0.05 ml of one of threeanti-VEGF medications (ranibizumab, bevacizumab, aflibercept) at thediscretion of the treating physician. Following 3 initial consecutivemonthly injections, re-injections were performed according to “treat andextend” protocol based on OCT findings. No attempt was made tostandardize injection interval between groups. Anesthesia depended onsubject and physician preference, and methods included topicalproparacaine or tetracaine drops, 2% lidocaine gel or subconjunctivalinjection of 2% lidocaine solution. Pre-injection prep varied withphysician preference. In PARA eyes eyelids were prepped with 10%betadine swab and additional betadine instilled into the conjunctiva. Innon-PARA eyes, no eyelid prep was performed and betadine was applied tothe conjunctiva using a 10% betadine swab immediately prior toinjection. Masking was not used routinely in either group. Injection wasdelivered by 30 g needles through the pars plana in either thesuperotemporal or inferotemporal quadrant according to physicianpreference. A cotton tipped applicator was placed over the injectionsite. Paracentesis was performed at the temporal limbus immediatelyafter injection using a 30 g needle on a tuberculin syringe from whichthe plunger had been removed. Following injection, all eyes wereirrigated with balanced salt solution. Between 2008 and 2013 topicalantibiotics were prescribed in both groups, and thereafter noprophylactic antibiotics were used.

Statistical Analysis

Data were summarized using means with standard deviations (continuousdata) or percentages (categorical data). Baseline characteristics werecompared between the PARA group (group 1), the non-PARA group (group 2),and the control group (group 3) using analysis of variance (ANOVA) orChi-square test, as appropriate. Post injection outcomes were evaluatedby comparing maximum post injection IOP, change in IOP from baseline topost injection, final IOP, change in RNFL thinning or loss and rate ofchange in RNFL thinning or loss between the treatment groups usingANOVA. Pearson's correlation coefficient was used to measure the extentof correlation between the IOP outcomes, change in RNFL thinning or lossand rate change in RNFL thinning or loss. SAS version 9.3 was used forstatistical analysis, all tests were two-sided, and p-values<0.05 wereconsidered significant. Multiple linear regression adjusting for age,gender, number of injections, follow-up time, baseline RNFL thinning orloss, and baseline IOP was used to determine if differences betweengroups remained after accounting for potential biases.

The results are now described.

Results

One hundred seventy nine eyes of 153 subjects who met the entry criteriawere included. Baseline characteristics are presented in FIG. 1.Seventy-five eyes received PARA (group 1) and 104 eyes did not (group2). An additional 44 untreated fellow eyes served as controls (group 3).Mean age was 81.0 and 79.0 years in groups 1 and 2 respectively. Fortypercent of subjects in group 1 and 42% in group 2 were male. Mean numberof injections was 9.9 (group 1) and 12.4 (group 2). Glaucoma, defined bypresence of EMR diagnosis of glaucoma and either use of topicalanti-ocular hypertensive drops or history of filtering procedure, waspresent in 20% (15 eyes) of group 1, 13% (13 eyes) of group 2 (p=0.147)and in 25% (11 eyes) controls. Of these, 11% (8 group 1 eyes) and 7% (7group 2 eyes) were taking multiple anti-ocular hypertensive drops. Atbaseline mean IOP was 16.0 mmhg (group 1) and 14.2 mmhg (group 2).Baseline mean RNFL thinning or loss was 87.8 um in group 1 and 88.6 umin group 2. There were no statistically significant differences amongbaseline parameters.

FIG. 2 presents post injection data. Mean follow-up was 27.7 and 31.3months in groups 1 and 2 respectively. Mean maxIOP was 19.1 mm hg and54.1 mm hg in groups 1 and 2 respectively (p<0.0001). Mean chgIOP was4.5 mm hg (range −15 to 24) after paracentesis and 39.9 mm hg (range 13to 60) in non-paracentesis eyes (p<0.0001). Mean chgRNFL thinning orloss was −1.3 um (group 1), −5.1 um (group 2) (p<0.0001) and −0.9 um(group 3). Mean pre-injection IOP at final visit (final IOP) were 14.5and 14.0 in groups 1 and 2 respectively. There was no statisticallysignificant change in pre-injection IOP between initial and final visitsin either group. Among eyes without glaucoma, mean chgRNFL thinning orloss was −1.0 um and −5.1 um in groups 1 and 2 respectively (p<0.0001)and −0.3 um in controls. Mean chgRNFL thinning or loss was −2.6 um(group 1), −5.0 um (group 2) (p=0.392) and −2.6 um (control) inglaucomatous eyes. Among glaucomatous eyes taking multiple drops,chgRNFL thinning or loss was −3.0, −9.6, −1.0 um in groups 1, 2 andcontrols respectively (p=0.017). Neither chgIOP (r=−0.175,p-value=0.121), nor maxIOP (r=−0.109, p-value=0.382) was significantlycorrelated with chgRNFLT.

Similar results were found when using multiple linear regressionaccounting for age, gender, number of injections, follow-up time,baseline RNFL thinning or loss, and baseline IOP. In these models,maxIOP, chgIOP, and chgRNFL all remained significant (p<0.0001 for each)and final IOP remained not significant (p=0.173). We checked all of theinteractions between baseline factors and confirmed that their inclusionin the models do not change the results presented.

FIG. 3 provides rate of RNFL thinning or loss change. Mean rate chgRNFLthinning or loss was −1.1 um/year vs. −3.3 um/year in groups 1 and 2respectively (p=0.0024). Subgroup analysis of eyes without glaucomarevealed mean rate chgRNFL thinning or loss was −0.88 um/year vs −3.54um/year in groups 1 and 2 respectively (p=0.0014). Among allglaucomatous eyes, mean rate chgRNFL thinning or loss of −1.99 um/year,−1.65 um/year and −1.45 um/year in groups 1 and 2 and controlsrespectively (p=0.938) and in those taking multiple drops it was −1.39um/year vs. −3.39 um/year in groups 1 and 2 (p=0.465). Standarddeviations were included with means to provide a sense of the underlyingdistributions. The distribution of change in RNFL and rate of change inRNFL resemble a bell shaped curve. If outliers are defined as +/−4standard deviations from the mean, there were no outliers for change inRNFL. For rate of change in RNFL, there were 3 outliers. A sensitivityanalysis was conducted by removing the outliers and comparing theresults to those in FIG. 3. This sensitivity analysis revealed nochanges to the results in FIG. 3, suggesting that the outliers are notaffecting the results presented.

Two eyes developed a single episode of pre-injection IOP>25 during thecourse of therapy. Endophthalmitis requiring vitreous tap andintravitreal antibiotic injection developed in 2 group 2 eyes (2subjects) (0.16% of injections, 1.9% of subjects). Both subjectsreceived prophylactic topical antibiotics after anti-VEGF injection(erythromycin ointment TID, ofloxacin drops QID). No paracentesis eyedeveloped hyphema or iris or lenticular injury.

Studies of the effect of IVAV on RNFL thinning or loss have reachedconflicting conclusions. In a prospective study of 49 eyes followed for12 months, Martinez-de-la Casa et al found intravitreal ranibizumabinjections used to treat AMD caused significant RNFL thinning or lossthinning (Martinez-de-la-Casa et al., Invest Ophthalmol Vis Sci. (2012)53(10):6214-6218). Three smaller retrospective series, two of which usedtime domain rather than spectral domain OCT, did not detect RNFLthinning or loss (Sobaci et al., Int J Ophthalmol. 2013; 6(2):211-215;Demirel et al., Curr Eye Res. 2015; 40(1):87-92; Horsley et al., Am JOphthalmol 2010; 150:558-561). The data demonstrates a statisticallysignificant loss of RNFL thinning or loss in non-glaucomatous eyes thatreceived IVAV without paracentesis.

Previous studies have reported intermittent or sustained elevation ofbaseline IOP in 3-6% of eyes during the course of treatment with IVAV(Adelman et al., J Ocular Pharmacol Ther. 2010; 26(1):105-110; Good etal., Br J Ophthalmol. 2011; 95(8):1111-1114; Tseng et al., J Glaucoma.2012; 21(4):241-247; Bakri et al., Ophthalmology. 2014; 121:1102-1108).A significant difference between mean initial and final baseline IOPswas not identified. The mean follow-up is shorter and mean number ofinjections is smaller than those reported by Tseng (Tseng et al., JGlaucoma. 2012; 21(4):241-247), however, without being bound to aparticular theory, it does not appear that sustained elevation ofbaseline IOP was responsible for the RNFL thinning or loss observed inthis study.

Intravitreal injection of 0.05-0.10 ml volume causes transient increasedIOP (Falkenstein et al., Retina. 2007; 27(8):1044-1047; Kim et al., Am JOphthalmol. 2008; 146(6):930-934; Sharei et al., Eur J Ophthlamol. 2010;20(1):174-179; Gismondi et al., J Glaucoma. 2009; 18(9):658-661). Animalstudies have demonstrated reduction of blood flow and tissue oxygenationat intraocular pressures greater than 50 mm hg and ocular perfusionpressure lower than 30 mm hg (Shonat et al., Invest Ophthalmol Vis Sci.1992; 33(11):3174-3180) as well as retinal ganglion cell damage when IOPof 110 mm hg is sustained for 90 minutes (Leung et al., Br J Ophthalmol.2009; 93(7):964-968). Maximum change in IOP has been correlated withprogressive optic nerve cupping in rats (Chauhan et al.,InvestOphthalmol Vis Sci. 2002; 43 (9):2969-2976). In humans, ocularperfusion and ischemia depends in part upon IOP and may be modulated bysystemic vascular factors (Deb et al., Indian J Ophthalmol. 2014;62(9):917-922; Wang et al., Curr Eye Res. 2014; 9:1-9; Yip et al.,Invest Ophthalmol Vis Sci. 2011; 52(11):8186-8192). Mean maxIOP and meanchgIOP for group 2 eyes in this study was higher than what has beenreported previously (Kim et al., Am J Ophthalmol. 2008; 146(6):930-934;Sharei et al., Eur J Ophthlamol. 2010; 20(1):174-179; Gismondi et al., JGlaucoma. 2009; 18(9):658-661). Among recognized factors (Falkenstein etal., Retina. 2007; 27(8):1044-1047; Kim et al., Am J Ophthalmol. 2008;146(6):930-934; Sharei et al., Eur J Ophthlamol. 2010; 20(1):174-179;Gismondi et al., J Glaucoma. 2009; 18(9):658-661; Bakri et al., Eye.2009; 23(1):181-185) routine use of 30 g needles may have contributed.Nonetheless, neither chgIOP nor maxIOP was significantly correlated withchgRNFL thinning or loss.

VEGF-A is required for the maintenance of normal retinal ganglion cells(Nishijima et al., Am J Pathol. 2007; 171(1):53-67) and inhibition ofVEGF's maintenance effect has the potential to cause RNFL injury. RNFLthinning or loss in group 1 eyes treated with anti-VEGF agents andparacentesis did not differ from that observed in fellow eye controls.Although a crossover effect of systemically absorbed anti-VEGF agents infellow eyes cannot be excluded (Scartozzi et al., Eye 2009, 23:1229;Bakbak et al., J Ocul Pharmacol Ther. 2013, 29:728-32; Bakbak et al.,Oman J Ophthalmol. 2016, 9:44-8) and VEGF inhibition may account for themodest RNFL thinning or loss observed in both groups. However, theeffect of VEGF inhibition alone does not account for the significantdifference in chgRNFLT observed between PARA and non-PARA groups.

Transient tissue ischemia injury may be caused by ischemia-reperfusionand chronic intermittent hypoxia. Ischemia-reperfusion injury has beenmodeled in skin using intermittent mechanical compression simulatingdiabetic foot ulcers (Reid R R, et al. J Surg Res. 2004 January;116:172-80.31) and with vascular occlusion simulating transient ischemicattacks (TIA) (Lee et al., AJNR 2004 25:1342-7). Sleep apnea has beenassociated with RNFL loss (Ferrandez et al. BMC Ophthalmol. 2016, 16:40;Zhao et al. J Glaucoma 2016; 25:e413-8).). Chronic intermittent hypoxiahas been modeled using brief periods of hypoxia simulating sleep apneain neural (Gozal D, et al. J Neuroscience, 2001, 21: 2442-50; Guo etal., Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 2016, 51:282-532)and cardiac (Park et al. Appl Physiol. 2007; 102(5):1806-14) tissues.

Without being bound to a particular theory, it is believed thatimmediate post injection IOP elevation in non-PARA eyes reducesperfusion and causes a transient period of tissue ischemia. As IOPnormalizes, reperfusion occurs. Tissue damage occurs as blood supply isre-established after a period of hypoxia or ischemia. VEGF confersneuroprotection (Sun et al., J Clin Invest. 2003; 111(12):1843-1851;Silverman et al., Neuroscience. 1999; 90:1529-1541; Jin et al., PNAS2000; 97: 10242-10247; Matsuzaki et al., FASEB J. 2001; 15:1218-1220)and has been shown to reduce infarct size (Dzietko et al., Transl.Stroke Res. 2013, 4:189-200; Zhang, et al., J. Clin. Investig. 2000,106:829-838; Zhang et al., J. Cerebr. Blood Flow Metab. 2002,22:379-392; Zhang, et al., Mol. Med. Rep. 2012, 6:1315-18) and apoptosis(Zhang, et al., Mol. Med. Rep. 2012, 6:1315-18) and increaseneurogenesis after stroke (Yang et al., Neuromol. Med. 2014,16:376-8843). VEGF also protects retinal ganglia fromischemia-reperfusion injury (Nishijima et al., Am J Pathol. 2007;171(1):53-67; Hirooka et al., Invest Ophthalmol Vis Sci. 2006;47(4):1653-1657). VEGF-A blockade exacerbates ganglion cell death due toocular hypertension (Foxton et al., Am J Pathol 2013; 182(4):1379-90)and increases infarct volume following focal transient cerebral ischemia(Bao et al., Zhongguo Yao Li Xue Bao. 1999, 20:313-8). In this regard,chronic VEGF inhibition intended to modulate detrimental angiogenic andvasopermiability effects may also chronically compromise VEGF'sphysiologic neuroprotective function and limit the ability of the RNFLto tolerate even brief episodes of transient ischemia-reperfusionassociated with IVAV.

Contrary to what was expected, non-PARA eyes with glaucoma did notdevelop significant RNFL thinning. All were being treated with topicalocular anti-hypertensive agents. The neuroprotective effects of topicalocular anti-hypertensive drops (Shih et al., Expert Rev Ophthalmol.2012; 7(2): 161-175) may have compensated for the loss of VEGF-derivedneuroprotection or acted synergistically with residual VEGF and therebylimited RNFL thinning or loss in these eyes.

Chronic VEGF inhibition due to IVAV, periocular injection, systemicadministration or topical administration may also limit the ability ofthe RNFL to tolerate transient ischemia-reperfusion injury. Further,because VEGF enters the systemic circulation following IVAV and reducesystemic anti-VEGF levels (Hård et al., Acta Paediatr, 2011, 100:1523-7;Avery et al., Ophthalmology, 2006 113:1695; Qian et al., Retina, 2011,31:161-8) they may render other tissues and organs normally protected byVEGF (Sun Y et al. J Clin Invest. 2003; 111(12):1843-51; Silverman, W Fet al. Neuroscience. 1999; 90:1529-41; Jin K L et al. PNAS 2000; 97:10242-7; Matsuzaki H et al. FASEB J. 2001; 15:1218-20; Luo Z, et al. AnnThorac Surg. 1997; 64(4):993-8; Tsurui Y et al. Transplantation. 2005May 15; 79(9):1110-5.) including the heart, brain and liver, morevulnerable to ischemia reperfusion injury initiated by other mechanisms.These include cardiac arrest (Crippen, SAS, 2005), cardiovascular shock,systemic hypotension, hypoxia, hypoxemia, hypovolemia, sleep apnea(Gozal D et al. J Neuroscience, 1 Apr. 2001, 21(7): 2442-50)transplantation surgery. (Lemasters, Annu Rev Pharmacol Toxicol, 1997,37:327-38), TIA (Lee S-K, et al. AJNR 2004 25: 1342-7) stroke, braintrauma and chronic pressure wounds (Mustoe, Am J Surg, 2004,187:65S-70S.) Therefore, supplemental neuroprotection may be consideredin persons taking anti-VEGF treatment, especially those who have ordevelop a condition conducive to producing ischemia reperfusion injury.

It is well known that acute cessation of circulation may cause tissueischemia and infarct, as occurs in retinal artery occlusion, ischemicoptic neuropathy and stroke. The data herein provide evidence to supportthat brief intermittent ischemic episodes caused by repeated andtransient circulatory compromise may culminate in tissue injuryincluding RNFL loss, and that neuroprotective agents may be protective.Similarly, tissue injury results from the brief, intermittent ischemicepisodes known as chronic intermittent hypoxia which characterize sleepapnea. Sleep apnea (without associated VEGF suppression) has beenassociated with reduced retinal sensitivity and RNFL loss (Ferrandez Bet al., Invest Ophthalmol Vis Sci 2014; 55:71119-25; Ferrandez B et al.BMC Ophthalmol. 2016, 16:40; Zhao et al. J Glaucoma 2016; 25:e413-8)apoptosis in the brain (Xu W, et al., Neuroscience. 2004;126(2):313-23), heart failure (Lavie L et al., Eur Respir J. 2009;33:1467-84. Constanzo M R et al. J AM Coll Cardiol 2015; 65:72-84;Khayat R et al., Heart Fail Rev 2009; 14:143-53) and sudden cardiacdeath due to arrhythmia (Gami A S et al., J AM Coll Cardiol 2013;62:610-6). The RNFL, a proxy for CNS neuronal tissue, was significantlythinner among newly diagnosed persons with sleep apnea compared tocontrol and also demonstrated significant progressive thinning in spiteof treatment with continuous positive airway pressure devices (CPAP)(Zengin M O et al., Int J Ophthalmol 2014; 7:704-8.). VEGF is elevatedin sleep apnea and although it may be involved in the pathogenesis ofsome of its complications (Ma J et al., J Huazhong Univ Sci TechnologMed Sci 2007; 27:157-60), VEGF is a hypoxia induced neurotropic factor(Wick A et al. J Neurosci 2002; 22:6401-7; Mu D et al. Neurobiol DIs2003; 14:524-34) that may facilitate an adaptive response to nocturnalhypoxemia (Schultz R et al., Am J Respir Crit Care Med 2002; 165:67-70;Lavie L et al., Am J Respir Crit Care Med. 2002 Jun. 15; 165(12):1624-8;Bernaudin M et al., J Cereb Blood FLow Metab 2002; 22:393-403.) andstroke (Mu D et al. Neurobiol DIs 2003; 14:524-34). Chronic VEGFinhibition during IVAV or systemic anti-VEGF administration may decreaseresistance to chronic intermittent hypoxemia (Wick A et al., JNeuroscience 2002; 22:6401-7) and thereby increase risk of consequencesof sleep apnea including RNFL loss and CNS neurodegenerative disorders.Therapeutic neuroprotection may be of importance for all individualswith sleep apnea including those treated with anti-VEGF agents. Further,neuroprotective compounds may prevent or ameliorate injury due tochronic intermittent hypoxia (Gozal D, et al. J Neuroscience, 2001, 21:2442-50; Guo et al., Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi.2016, 51:282-532; Ferrandez et al. BMC Ophthalmol. 2016, 16:40; Zhao etal. J Glaucoma 2016; 25:e413-8) and or ischemia-reperfusion injurycaused by conditions including but not limited to TIA (Lee et al., AJNR2004 25:1342-7) even among those treated with CPAP and/or in the absenceof iatrogenic VEGF suppression.

Age-related decline in RNFL thickness has been reported to range from0.16 to 0.5 um per year, and is greater in persons 50 years of age andolder (Parikh et al., Ophthalmology. 2007; 114(5):921-926; Wang et al.,The Beijing Eye Study 2011. PLOS One Jun. 4, 2013). While annual RNFLthinning or loss in PARA and control groups among all eyes andnon-glaucomatous eyes was similar to that reported for persons over 50years (Wang et al., The Beijing Eye Study 2011. PLOS One Jun. 4, 2013),it was 3 to 4 times greater in the corresponding non-PARA groups. Amongglaucoma suspects, a lum per year more rapid annual decline in RNFLthickness corresponded to a 2.05-times higher risk of developing avisual field defect (Miki et al., Ophthalmology. 2014. 121:1350-8).Further, RNFL thinning or loss has been associated with glaucomaassociated disability and reduction in quality of life (Gracitelli etal., JAMA Ophthalmol. 2015, 133:384-90).

While annual RNFL loss in PARA and control groups among all eyes andnon-glaucomatous eyes was similar to that reported for persons over 50years (Wang et al., The Beijing Eye Study 2011. PLOS One, 2013), it was3 to 4 times greater in the corresponding non-PARA groups. Glaucomatouseyes have an abnormal optic nerve blood-flow (Flammer et al., Prog RetinEye Res. 1998, 17:267-89) and those with advanced disease may be moresusceptible to ischemia-reperfusion injury. A statistically significanteffect of paracentesis was not detected in eyes with advanced glaucomain this study, but the number of eyes was small. Further study of thissubgroup is warranted.

In summary, the results presented herein provide evidence that IOPelevations of short duration in the setting of chronic VEGF suppressionmay be deleterious to the RNFL, especially in eyes not being treated forglaucoma. Clinical significance may depend upon duration of an anti-VEGFtherapy such as IVAV as well as comorbidities that may compromise theRNFL including advanced glaucoma and ocular or systemic disease ordisorders which predispose to chronic intermittent hypoxia and/orischemia-reperfusion injury. Future investigation of the clinicalsignificance of our observations would be especially relevant in eyesfor which the duration of treatment and total number of injections mayexceed those reported or for those who have ocular or systemiccomorbidities. The potential mechanism and role for neuroprotectiveagents in limiting RNFL thinning or loss during IVAV or other anti-VEGFtherapy warrants exploration.

Example 2: Use of Tafluprost, an Anti-Pcular Hypertension Drop withNeuroprotective Properties, to Decrease RNFL Thinning or Loss andIncrease the Interval Between IVAV Injections, in Eyes Treated with IVAV

Intravitreal anti-VEGF injections (IVAV) are the standard of care forthe treatment of choroidal neovascularization and macular edema due to avariety of disorders including macular degeneration and diabeticretinopathy, both leading causes of blindness in the western world. IVAVcauses an immediate, transient elevation of intraocular pressure (IOP)which temporarily compromises intraocular circulation. VEGF suppressionintended to modulate detrimental angiogenic and vasopermiability effectsalso chronically compromises VEGF's physiologic neuroprotective functionrendering eyes more susceptible to ischemia-reperfusion injury. Multipleepisodes of ischemia-reperfusion injury unmitigated by innateVEGF-derived neuroprotection may culminate in RNFL thinning or loss.Without wishing to be bound by a particular theory, it is believed thatdaily use of glaucoma drops with neuroprotective properties may reduceor prevent RNFL thinning or loss associated with IVAV and also increaseinterval between treatments.

Intravitreal injection of 0.05-0.10 ml volume transiently elevates IOP(Falkenstein et al., Retina 2007; 27:1044-7; Kim et al., Am J Ophthalmic2008; 146:930-4; Sharei et al., Eur J Ophthalmic 2010; 20:174-9;Gismondi et al., J Glaucoma 2009; 18:658-61). IOP elevation with IVAVmay reach levels that have been sufficient to compromise ocular bloodflow and tissue oxygenation in animals (Shonat et al., Invest OphthalmolVis Sci 1992l 33:3174-80), and to cause visible central retinal arterialpulsation in humans (Nazari et al., Iranian J Ophthalmology 200921:13-18). The retinal artery pulsations, which indicate intraocularpressure exceed intra-arterial pressure and that retinal circulation iscompromised, may last from 30 seconds to 8 minutes (Nazari et al.,Iranian J Ophthalmology 2009 21:13-18). IOP is highest immediately afterinjection. As it normalizes and retinal circulation is reestablished, inmost instances within 30 minutes, the ischemia-reperfusion cycle occurs.

Data in FIG. 2 demonstrates a statistically significant difference inboth maximum post injection IOP (maxIOP) and magnitude change in IOPfrom pre injection baseline (chgIOP) in eyes undergoing IVAV with andwithout adjunctive paracentesis (PARA). Higher IOP in non-PARA eyes wascaused by intravitreal injection volume. PARA immediately normalizedintraocular volume and consequently prevented post injection IOP rise. Astatistically significant change in RNFL thinning or loss thickness(chgRNFL thinning or loss) was also detected (greater RNFL thinning orloss in nonPARA eyes) (FIG. 2). However, neither chgIOP (r=−0.175,p-value=0.121) nor maxIOP (r=−0.109, p-value=0.382) was significantlycorrelated with chgRNFL thinning or loss (FIG. 4). Similar results werefound when using multiple linear regression accounting for age, gender,number of injections, follow-up time, baseline RNFL thinning or loss,and baseline IOP. In these models, maxIOP, chgIOP, and chgRNFL allremained significant (p<0.0001 for each) and final IOP remained notsignificant (p=0.173). These data suggest that elevated IOP alone isinsufficient to account for the observed RNFL thinning or loss. Itfollows that control of the immediate post injection IOP with a singledose of anti-glaucoma medication prior to IVAV as has been suggested(Kim) would not be likely to prevent RNFL thinning or loss.

Subgroup analysis revealed that the significant decrease in RNFLthinning or loss in nonPARA eyes developed only in non-glaucomatouseyes. RNFL thinning was larger in magnitude (FIG. 2), and progressed ata statistically significantly greater rate in non-glaucomatous eyes(FIG. 3). This paradox, that eyes with glaucoma, a disease characterizedby progressive RNFL thinning or loss thinning in the setting of elevatedIOP, are less likely to develop RNFL thinning or loss during IVAV, maybe understood upon considering the following. First,ischemia-reperfusion is an established cause of neurologic injury(Winquist and Kerr, Neurology 1997; 49 (Suppl 4) S23-6) and second, itmay be mitigated by endogenous VEGF (Nisijima et al., Am J Pathol 2007;171:53-67) or exogenous therapeutic agents which conferneuroprotectionith against ischemia-reperfusion injury.

VEGF is required for the maintenance of normal retinal ganglion cells(Nisijima et al., Am J Pathol 2007; 171:53-67) and inhibition of VEGF'smaintenance effect has the potential to cause RNFL injury. Further, VEGFnormally protects against ischemia-reperfusion injury (Nisijima et al.,Am J Pathol 2007; 171:53-67). The data in FIG. 2 shows that chgRNFLthinning or loss in eyes treated with anti-VEGF agents plus paracentesisdid not differ from that observed in fellow eye controls. This suggestsVEGF inhibition itself does not account for the significant differencein chgRNFL thinning or loss we observed between PARA and non-PARAgroups. Taken as a whole, the data in the tables suggest RNFL thinningor loss required that two factors were present, that is, it developed ineyes exposed to repeated episodes of ischemia-reperfusion while theywere also vulnerable to ischemic injury owing to compromised innateneuroprotiction during in a state of deficient innate chronic iatrogenicVEGF suppression.

Glaucoma drops are administered to control elevated IOP. Some also havedemonstrable neuroprotective properties. For example, in animals,tafluprost protects retinal gangion cells from apoptosis both in vitroand in vivo (Kanamori et al., Graefes Arch Clin Exp Ophthalmol 2009;247:1353-60). Further, prostaglandin F2α analogues including tafluprostimproved optic nerve blood flow in animal models (Kurishima et al., ExpEye Res 2010; 91:853-9). Daily topical administration of tafluprost wasalso shown to be protective against endothelin 1 induced ischemicretinal injury in animals. RNFL thinning or loss was thicker in treatedeyes (Nagata et al., Invest Ophthalmol Vis Sci 2014 55:1040-7). Theseanimal studies of ischemic injury to the optic nerve and retinademonstrate neuroprotective properties of glaucoma medications in thesetting of ischemia, and may be relevant to ischemia-reperfusion injuryassociated with IVAV.

Among non-glaucomatous eyes there was a statistically significantdifference in chgRNFL thinning or loss between PARA and nonPARA eyes.This suggests non-glaucomatous nonPARA eyes subject to elevated IOP andrepeated ischemia-reperfusion episodes in the setting of reduced orabsent innate VEGF suffered loss of RNFL thinning or loss. In contrast,no significant difference in chgRNFL thinning or loss between PARA andnon-PARA eyes was detected in the glaucoma eye subgroup (eyes usingdaily glaucoma drops) (FIG. 2) and RNFL thinning or loss was similar intreatment eyes and controls. These data support the hypothesis thatglaucoma drops used on a daily basis during IVAV provided pharmacologicneuroprotection against ischemia-reperfusion injury. This pharmacologicprotection prevented statistically significant RNFL thinning or loss innon-PARA eyes.

There is a second potential beneficial effect of daily use of theprostaglandin F2α analogue tafluprost during the course of IVAV. Theneuroprotective effect of tafluprost is mediated by inhibition ofendothelin 1 (Nagata et al., Invest Ophthalmol Vis Sci 2014 55:1040-7).Down-regulation of endothelin 1, a stimulator of VEGF expression,decreases VEGF mRNA production (Tsuda et al., J Glaucoma 2013;22:389-403). It follows that daily use may also decrease intraocularVEGF concentration, and thereby potentiate anti-VEGF agents used inIVAV. Daily topical use may be expected to increase interval betweenanti-VEGF injections and therefore decrease the number and frequency ofanti-VEGF injections required per patient in a treat and extend protocolcompared to control. Sustained IOP elevation is related to number ofinjections (Abedi et al., Semin Ophthamol, 2013, 28:126-130; Singh andKim, Drugs Aging, 2012, 28:949-956).

The annual RNFL thinning or loss reported herein was 3 to 4 timesgreater in the non-glaucomatous non-PARA eyes than that normallyattributed to aging (Parikh et al., Ophthalmology. 2007; 114(5):921-6;Wang et al., PLOS One, 2013, 0066763). In a study of glaucoma suspects,a 1 um per year more rapid annual decline in RNFL thinning or losscorresponded to a 2.05-times higher risk of developing a visual fielddefect (Miki et al., Ophthalmology. 2014; 121(7):1350-8). Further, RNFLthinning or loss has been associated with glaucoma associated disabilityand reduction in quality of life (Gracitelli et al., JAMA Ophthalmol.2015; 133(4):384-90). The clinical significance of IVAV associated RNFLthinning or loss thinning may depend upon additional factors.Susceptibility to ischemia is related to ocular perfusion which may becompromised by systemic vascular factors including diabetes (Panes etal., Circulation 1996; 13: 161-7), hypertension and anti-hypertensivemedications (Deb et al., Indian J Ophthalmol. 2014; 62(9):917-22; Wanget al., Curr Eye Res. 2014; 9:1-9; Yip et al., Invest Ophthalmol VisSci. 2011; 52(11):8186-92). It would seem prudent therefore to takemeasures to minimize RNFL thinning or loss thinning, especially inpersons with systemic vascular disease, in those who are younger orwhose duration of treatment and total number of injections is expectedto be greater.

Without wishing to be bound by a particular theory, it is believed thattafluprost may decrease or prevent RNFL thinning that develops duringIVAV when adjunctive paracentesis is not performed, and that treatmentwith tafluprost will allow an increase the interval between anti-VEGFinjections and therefore result in a decreased number and/or frequencyof anti-VEGF injections per subject in a treat and extend protocolcompared to control.

To demonstrate tafluprost decreases RNFL thinning in eyes withoutglaucoma that are being treated with IVAV, and that treatment withtafluprost can further increase the interval of time between multipleIVAV procedures and/or reduce the number of IVAV procedures necessaryfor an effective IVAV treatment regimen, intraviteal injections areperformed according to a treat and extend protocol (Spaide, Am JOphthalmol. 2007, 143:679-80; Engelbert et al., Retina, 2010,30:1368-75; Engelbert et al., Retina, 2009, 29:1424-31) in three groups.The one treatment group (Groups 1) receives tafluprost (below), whilethe control group (Group 2) receives placebo (artificial tears). Group1: Tafluprost 0.0015% one drop once a day at bedtime for the duration ofIVAV. Group 2: Control, placebo (preservative free artificial tears) onedrop once a day at bedtime for the duration of IVAV.

To determine if treatment with tafluprost increases the interval betweenintravitreal anti-VEGF injections and thereby decreases the number ofinjections required in a treat and extend protocol, total number ofinjections/year and intervals between injections are compared betweenthe treatment groups and the measures of change in RNFL thickness, rateof change in RNFL thickness, number of injections per year, intraocularpressure, and visual acuity are determined. Adverse events that aremonitored include endophthalmitis, anterior segment injury (hyphema,lens injury) and pre-injection IOP>25 mm hg.

Baseline refraction is performed. Ophthalmic photographers obtainedsd-OCT (Spectralis, Heidelberg Engineering) prior to each injection andscanned images into the electronic medical record. RNFL thicknesscalculated by the autosegmentation algorithm including mean central,inferior, superior, nasal and temporal thickness is recorded.Intraocular pressure is obtained using tonopen (Reichert Technologies)before and immediately and 10 minutes after injection. Tonopen is usedin lieu of applanation in order to facilitate immediate post-injectionIOP measurement. If the 10-minute post injection IOP was greater than 30mm hg, additional IOP readings were obtained at 10 minute intervalsuntil IOP was 30 mm hg or less.

Intravitreal injections are administered through 30 gauge needlesthrough the pars plana in either the superotemporal or inferotemporalquadrant according to physician preference. A cotton tipped applicatorwas placed over the injection site. One of three anti-VEGF medications(ranibizumab, bevacizumab, aflibercept) chosen at the discretion of thetreating physician is administered in 0.05 ml volume. Anesthesia ischosen based on subject and physician preference, and methods mayinclude topical proparacaine or tetracaine drops, 2% lidocaine gel orsubconjunctival injection of 2% lidocaine solution. Standardpre-injection sterilization will include eyelid prep using 10% betadineswab and additional betadine instilled into the conjunctiva. Betadine isapplied to the conjunctiva using a 10% betadine swab immediately priorto injection. Following injection, all eyes are irrigated with balancedsalt solution.

The methods and materials are now described.

Treat and Extend Protocol

Subjects are injected with intravitreal anti-VEGF once a month for threemonths. Once stability is achieved with monthly dosing, (stable visualacuity, an absence of macular hemorrhage, and a dry OCT) the subject isinstructed to return in six weeks. Visual acuity, clinical findings, andOCT changes are recorded again and subjects receive an injection. Theinterval to the next visit with scheduled injection is based on anobserved change in the above parameters. If there are no changes, theinterval to the next visit is extended to seven weeks with scheduledinjection. However, if there is evidence of disease reactivation, theinterval for the next scheduled injection and examination is shortenedby one week.

Topical Medication

Group 1 subjects will use tafluprost once daily at bedtime throughoutthe course of therapy. The control group (group 2) will receivepreservative free artificial tears once daily at bedtime throughout thecourse of therapy.

Statistical Analysis

Sample size was determined as a balance of statistical power andfeasibility of recruitment. To evaluate sample size, the size ofdetectable standard deviation unit (SDU) was used. The SDU correspondsto a beta coefficient in a regression model when we assume the standardnormal deviate (i.e. N(0,1)). A sample size of 65 per group (130 total)will have 80% power to detect an SDU of 0.50 (a moderate effect size).An SDU of 0.50 corresponds to an absolute difference equal to half thesize of the standard deviation that translates into a 13% shift in astandard normal distribution.

Data is summarized using means with standard deviations (continuousdata) or percentages (categorical data). Baseline characteristics (age,gender, refractive error, IOP and mean central RNFL) (Alasil et al., JGlaucoma 2013, 22:532-41) are compared between the groups using analysisof variance (ANOVA) or Chi-square test, as appropriate. Post injectionoutcomes are evaluated by comparing change in IOP from baseline to finalIOP, change in RNFL thinning or loss and rate of change in RNFL thinningor loss between the treatment groups using ANOVA. Pearson's correlationcoefficient is used to measure the extent of correlation between the IOPoutcomes, change in RNFL thinning or loss and rate change in RNFLthinning or loss. SAS version 9.3 is used for statistical analysis, alltests are two-sided, and p-values<0.05 are considered significant.Multiple linear regression adjusting for age, gender, refractive error,number of injections, follow-up time, baseline RNFL thinning or loss,and baseline IOP are used to determine if differences between groupsremained after accounting for potential biases.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

What is claimed is:
 1. A method of protecting against retinal nervefiber thinning or loss and ischemia-reperfusion injury associated withintravitreal anti-VEGF injection (IVAV) treatment in a subject in needthereof, said method comprising administering an effective amount of acomposition comprising a prostaglandin analog to the subject, whereinthe subject is a human subject not receiving a neuroprotectiveprostaglandin analog for the treatment of glaucoma, and wherein thesubject will experience or has experienced IVAV, wherein saidprostaglandin analog is tafluprost.
 2. The method of claim 1 whereinsaid composition is capable of crossing the blood-brain barrier and theblood-retina barrier.
 3. The method of claim 1, wherein said compositionis administered to said subject by way of an administration routeselected from the group consisting of topical, oral, intravenous,periocular injection and an intraocular implant drug delivery device. 4.The method of claim 1, wherein said composition is administered to saidsubject daily for at least three months.
 5. The method of claim 1,wherein said composition is administered to said subject by way of oneor more treatment regimen selected from the group consisting of at leastonce prior to IVAV treatment and at least once following IVAV treatment.6. The method of claim 1, wherein said composition is administered tosaid subject daily for the duration and for at least three monthsfollowing an IVAV treatment regimen.
 7. The method of claim 6, wherebyone or more of the time interval between IVAV injections is increasedand the number of IVAV injections is decreased relative to the averagetime interval and number of injections received respectively by controlindividuals.
 8. The method of claim 1 comprising the steps of: a)administering an effective amount of tafluprost to the subject; and b)administering an intravitreal anti-VEGF injection (IVAV) treatment tothe subject.
 9. The method of claim 8, wherein step a) and step b) areperformed in a manner selected from the group consisting of: step a) isperformed prior to step b), step b) is performed prior to step a), andstep a) is performed concurrently with step b).